F. tularensis, is a facultative intracellular bacterial pathogen considered as a major biological warfare agent, mainly because it is very infectious and can cause life threatening illness in humans. Without treatment, the mortality rate has been as high as 30 to 60% for the pulmonary form of tularemia, which is the most likely form in case of an act of bioterrorism. Treatment of human tularemia relies upon antibiotic therapies but relapse is frequent. Also, it is possible to engineer multiresistant strains. A live attenuated vaccine is available but confers incomplete protection, particularly if administered after exposure. Therefore, there is a crucial need for new treatments and vaccines against tularemia. However, understanding of the mechanisms of virulence in F. tularensis is currently too limited to address this problem efficiently. This is partly due to the lack of genetic tools, particularly a simple method of general mutagenesis. Transposable elements have become valuable mutagenic tools for genetic and molecular analysis in many different bacteria. However, no direct transposition has been observed so far in F. tularensis. The main goals of this proposal are as follows: first, to develop a simple and reproducible method of transposon mutagenesis in F. tularensis, by using a fully functional Tn5 transposition system (EZ::TN TM TransposomeTM), reconstituted in vitro. Our preliminary work has shown strong evidence for direct transposition in F. tularensis with this system; second, to perform transposon signature-tagged mutagenesis to isolate mutants attenuated in their virulence, in the mouse model. This pilot study should provide important informations on the genes involved in the virulence of F. tularensis and a better understanding of the pathogenicity of this microorganism. Some of the virulence genes will be used as potential targets for new approaches in drug design and vaccine development.